Persulfated oligosaccharide acting on selectins and chemokine

ABSTRACT

A persulfated saccharide compound capable of acting on selectin and chemokine. According to the present invention, there are provided a saccharide compound capable of interacting with L-selectin, P-selectin and chemokine; a pharmaceutical composition; and an agent for treating or preventing a disease of which sideration is associated with biological events mediated by any of L-selectin, P-selectin and chemokine.

TECHNICAL FIELD

The present invention relates to a persulfated oligosaccharide capableof acting on selectins and chemokines. Particularly, the presentinvention relates to a saccharide compound which interacts withL-selectin, P-selectin and chemokine which are an pro-inflammatorymolecule, as well as a therapeutic or prophylactic agent for a diseaseof which sideration is associated with biological events mediated by anyof L-selectin, P-selectin and chemokine. More particularly, the presentinvention relates to an agent for treating or preventing a disease ofwhich sideration is associated with biological events mediated withL-selectin, P-selectin, chemokine or the like; a lead compound for drugdiscovery for the therapeutic or prophylactic agent; a saccharidecompound useful for designing or the like of the lead compound, as wellas a therapeutic or prophylactic agent useful for treating or preventinga disease such as inflammatory disease, allergic disease, cancermetastasis, myocardial dysfunction and multiple organ failure.

BACKGROUND ART

It is known that inflammation is induced by an adhesion molecule onleukocytes and a molecule for promoting adhesion with a vascularendothelial cell, and that certain adhesion molecules are involved ininflammation. The adhesion molecule includes, for instance, a selectinfamily which is represented by L-selectin and P-selectin.

It is known that, in the interaction between the L-selectin orP-selectin and its ligand, sulfation of the ligand plays an importantrole. For example, it is known that tyrosine sulfation of P-selectinglycoprotein ligand-1 is necessary for the interaction between theP-selectin glycoprotein ligand-1 and any of L-selectin and P-selectin[Sako, D. et al., Cell, 83, 323-331 (1995); Pouyani, T. et al., Cell,83, 333-343 (1995); and Spertini, O. et al., J. Cell Biol., 135, 523-531(1996)].

In addition, it is known that a ligand of L-selectin on a highendothelial small vein binds to L-selectin in a sulfation-dependentmanner [Imai, Y. et al., Nature, 361, 555-557 (1993); Hiraoka, N. etal., Immunity, 11, 79-89 (1999); Bistrup, A. et al., J. Cell Biol., 145,899-910 (1999)]. Further, it is known that HNK-1 reactive sulfoglucronylglycolipid [Needham, L. K. et al., Proc. Natl. Acad. Sci. U.S.A., 90,1359-1363 (1993)], heparin oligosaccharide [Nelson, R. M. et al., Blood,11, 3253-3258 (1993)] and heparan sulfate glycosaminoglycan [Koenig, A.et al., J. Clin. Invest., 101, 877-889 (1998)] binds to any ofL-selectin and P-selectin.

However, since any of compounds which binds to any of L-selectin andP-selectin have a high molecular weight, under the circumstances, it isdifficult to design a lead compound capable of regulating a bindingbetween L-selectin, P-selectin or the like and a ligand and that amedicament using the compound has not yet been successfully developed.

DISCLOSURE OF INVENTION

An object of the present invention is to provide a saccharide compound,by which the regulation of binding between any of L-selectin, P-selectinand chemokine and its ligand, the regulation of biological eventsmediated by any of L-selectin, P-selectin and chemokine, improvement insymptom of a disease of which sideration is associated with thebiological events, and provision of a lead compound for a therapeutic ora prophylactic agent for the disease can be achieved. Also, an object ofthe present invention is to provide an agent for treating or preventinga disease of which sideration is associated with biological eventsmediated by any of L-selectin, P-selectin and chemokine, which canimprove symptom of a disease such as inflammatory disease, allergicdisease, cancer metastasis, myocardial disorder and multiple organfailure, and can exhibit high affinity in a living body.

Specifically, the present invention relates to[1] a saccharide compound represented by the general formula (I):

wherein R¹, R², R³ and R⁴ each independently represent a hydrogen atomor a sulfonic group, or the general formula (II):

wherein R⁵, R⁶, R⁷ and R⁸ each independently represent a hydrogen atomor a sulfonic group;[2] a saccharide compound represented by the general formula (III):

wherein R⁹ and R¹⁰ each independently represent a hydrogen atom or asulfonic group, and m is 3 or 4, or the general formula (IV):

wherein R¹¹ and R¹² each independently represent a hydrogen atom or asulfonic group, and n is 3 or 4;[3] a pharmaceutical composition comprising the saccharide compound ofthe above item [1] or [2] as an active ingredient; and[4] an agent for treating or preventing a disease of which sideration isassociated with biological events mediated by any of L-selectin,P-selectin and chemokine, wherein said agent comprises the saccharidecompound of the above item [1] or [2] as an active ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing the results of investigation byimmunoprecipitation for the effects of sulfation on the interactionbetween a chondroitin sulfate/dermatan sulfate chain in versican andeach of L-selectin, P-selectin and CD44.

FIG. 2 is a view showing the results of investigation by enzyme-linkedimmunosorbent assay for the effects of a persulfated CS/DS chain onbinding between versican and any of L-selectin, P-selectin and CD44. InFIG. 2, a cross shows keratan sulfate, an open triangle beingchondroitin, a open square being chondroitin sulfate A, a open circlebeing chondroitin polysulfate, a solid triangle being dermatan, a solidsquare being dermatan sulfate, a solid circle-solid line being dermatanpolysulfate, and a solid circle-dashed line being chondroitin sulfate E.In addition, in FIG. 2, panel A is the results for binding betweenversican and L-selectin, panel B being the results for binding betweenversican and P-selectin, and panel C being the results for bindingbetween versican and CD44.

FIG. 3 is a view showing a disaccharide composition of versican-derivedglycosaminoglycan. Panel A shows the results of analysis of adisaccharide composition of versican-derived glycosaminoglycan wastreated with chondroitinase ABC and then derivatized with2-aminobenzamide (2-AB). Panel B shows the results of analysis of adisaccharide composition of versican-derived glycosaminoglycan which wastreated with chondro-6-sulfatase and chondroitinase ABC and thenderivatized with 2-AB. In addition, in FIG. 3, 0S shows the elutionposition of 2-AB-derivatized ΔDi-0S, 4S being the elution position of2-AB-derivatized ΔDi-4S, 6S being the elution position of2-AB-derivatized ΔDi-6S, diS_(D) being the elution position of2-AB-derivatized ΔDi-di(2,6)S, diS_(E) being the elution position of2-AB-derivatized ΔDi-di(4,6)S, and UA2S being the elution position of2-AB-derivatized ΔDi-UA2S.

FIG. 4 is a view showing the results of investigation for the effects ofsulfation on the interaction between versican and chemokine. Panel Ashows the disaccharide composition of a product derivatized with2-aminobenzamide. 0S shows the elution position of 2-AB-derivatizedΔDi-0S, 4S being the elution position of 2-AB-derivatized ΔDi-4S, 6Sbeing the elution position of 2-AB-derivatized ΔDi-6S, diS_(D) being anelution position of 2-AB-derivatized ΔDi-di(2,6)S, and diS_(E) being anelution position of 2-AB-derivatized ΔDi-di(4,6)S. Panel B shows theresults of investigation by enzyme-linked immunosorbent assay for theeffects of sulfation on the interaction between versican and chemokine.BSA represents bovine serum albumin, 2B1 being anti-versican monoclonalantibody 2B1, L-Ig being L-selectin-Ig, E-Ig being E-selectin-Ig, P-Igbeing P-selectin-Ig, SLC being secondary lymphoid tissue chemokine,SLC-T being C-terminal truncated secondary lymphoid tissue chemokine,IP-10 being γ-interferon inducible protein-10, PF4 being platelet factor4, SDF-1β being stromal cell-derived factor-1β, and SDF-1α being stromalcell-derived factor-1α. Each of the bars in panel B shows mean±standarddeviation from tetraplicate measurement. A black bar is the results foruntreated conditioned medium-derived versican, and a hatched bar beingthe results for sodium chlorate-treated conditioned medium-derivedversican. Each of the bars shows mean±standard deviation fromtetraplicate measurement.

FIG. 5 is a view showing the results of investigation by enzyme-linkedimmunosorbent assay for the effects of a persulfated CS/DS chain onbinding between versican and chemokine. Expression of an abscissadenotes the same as that of FIG. 4. In addition, in each of lanes, bar 1shows the results in the absence of glycosaminoglycan, bar 2 beingchondroitin, bar 3 being chondroitin sulfate A, bar 3 being chondroitinpolysulfate, bar 5 being dermatan polysulfate, and bar 6 beingchondroitin sulfate E. Each of the bars shows mean±standard deviationfrom triplicate measurement.

FIG. 6 shows the sensorgram of BIAcore, in which the interaction betweenimmobilized glycosaminoglycan and each of chemokine, L-selectin and CD44is recorded. In FIG. 6, SLC represents secondary lymphoid tissuechemokine, IP-10 being γ-interferon inducible protein-10, SDF-1β beingstromal cell-derived factor-1β, CS E being chondroitin sulfate E, and CSA being chondroitin sulfate A. In FIG. 6, the response in a resonanceunit is recorded as a function of time.

FIG. 7 is a view showing the results of identification of the structureof a fragment which interacts with each of L-selectin, P-selectin andchemokine. Panel A shows HPLC chromatogram of a hyaluronidase-digestedproduct of each of chondroitin sulfate A (CS A in the figure),chondroitin sulfate C(CS C in the figure) and chondroitin sulfate E (CSE in the figure). Panel B shows a disaccharide composition of each ofthe fraction a, the fraction c, the fraction e-1 and the fraction e-2.Panel C is a schematic view of the structure of each of the fraction a,the fraction c, the fraction e-1 and the fraction e-2, a solid triangleshows GlcA, a hatched circle being GalNAc, 4S being 4-O-sulfation, 6Sbeing 6-O-sulfation, β3 being β1-3 linkage, and β4 being β1-4 linkage.Panel D shows results of investigation for the interaction between anoligosaccharide contained in each of the fraction a, the fraction c, thefraction e-1 and the fraction e-2, and each of L-selectin, P-selectin,CD44 and chemokine. The expression of an abscissa denotes the same asthat of FIG. 4. In addition, in each of lanes, bar 1 shows the resultsof the case where streptoavidin-conjugated alkaline phosphatase is used,bar 2 being the results of the case where streptoavidin-conjugatedalkaline phosphatase coupled to biotinylated fraction a is used, bar 3being the results of the case where streptoavidin-conjugated alkalinephosphatase coupled to biotinylated fraction c is used, bar 4 being theresults of the case where streptoavidin-conjugated alkaline phosphatasecoupled to biotinylated fraction e-1 is used, and bar 5 being theresults of the case where streptoavidin-conjugated alkaline phosphatasecoupled to biotinylated fraction e-2 is used. Each of the bars denotesmean±standard deviation from tetraplicate measurement.

FIG. 8 is a view showing the results of investigation for the effects ofa persulfated CS/DS chain on chemokine activity. In FIG. 8, SLCrepresents secondary lymphoid tissue chemokine, SLC-T being C-terminaltruncated secondary lymphoid tissue chemokine, CS E being chondroitinsulfate E, and CS A being chondroitin sulfate A. In addition, in FIG. 8,the arrowhead shows the time point at which stimulation was given.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is based on surprising findings that a saccharidecompound comprising, as a constituent unit, a disaccharide moiety of thegeneral formula (III′):

wherein R¹³ and R¹⁴ each independently represent a hydrogen atom or asulfonic group, and k is an arbitrary natural number, ora disaccharide moiety of the general formula (IV′):

wherein R¹⁵ and R¹⁶ each independently represent independently ahydrogen atom or a sulfonic group, and l is an arbitrary natural number,among sulfated glycosaminoglycans, in particular, a tetrasaccharidecompound having a repeat unit of GlcAβ1-3GalNAc(4,6-O-disulfate) or atetrasaccharide compound having a repeat unit ofIdoAα1-3GalNAc(4,6-O-disulfate) interacts with each of L-selectin,P-selectin and chemokine, and surprising findings that thetetrasaccharide compounds inhibit physiological activities of chemokine.

In the specification, “GlcA” represents D-glucronate residue, “GalNAc”being N-acetyl-D-glactosamine residue, “IdoA” being L-iduronate residue,and “HexA” being hexuronate residue. In addition, “β1-3” means β1-3linkage, and “α1-3” means α1-3 linkage.

The saccharide compound of the present invention includes a saccharidecompound represented by the general formula (I):

wherein R¹, R², R³ and R⁴ each independently represent a hydrogen atomor a sulfonic group, or

a saccharide compound represented by the general formula (II):

wherein R⁵, R⁶, R⁷ and R⁸ each independently represent a hydrogen atomor a sulfonic group, and

a saccharide compound represented by the general formula (III):

wherein R⁹ and R¹⁰ each independently represent a hydrogen atom or asulfonic group, and m is 3 or 4, or

a saccharide compound represented by the general formula (IV):

wherein R¹¹ and R¹² each independently represent a hydrogen atom or asulfonic group, and n is 3 or 4. Although the saccharide compound of thepresent invention is a compound comprising one of a tetra- tooctasaccharide, there are exhibited excellent effects that thesaccharide compound interacts with any of L-selectin, P-selectin andchemokine, to regulate physiological functions associated with any ofL-selectin, P-selectin and chemokine. In addition, the saccharidecompound of the present invention can be simply prepared uponpreparation thereof. Further, since the saccharide compound of thepresent invention is a compound comprising one of a tetra- tooctasaccharide, there are exhibited excellent effects that it can beused as a low molecular compound capable of regulating binding betweenat least one kind selected from the group consisting of L-selectin,P-selectin and chemokine, and the ligand thereof, or as a basis fordesigning lead compound thereof. In addition, according to thesaccharide compound of the present invention, there can be carried outthe regulation of binding between any of L-selectin, P-selectin andchemokine, and the ligand thereof, the regulation of biological eventsmediated by any of L-selectin, P-selectin and chemokine, the improvementof symptom of a disease of which sideration is associated with thebiological events, and the provision of a lead compound for a remedy oran inventive for the disease.

In the present invention, from the viewpoint of a molecular weight,particularly, a saccharide compound represented by the above generalformula (I) or general formula (II) is preferable.

In the above general formula (I), R¹, R², R³ and R⁴ each independentlyrepresent a hydrogen atom or a sulfonic group. In addition, the sulfonicgroup may have a substituent, as long as the object of the presentinvention is not hindered. A saccharide compound in which any of R¹, R²,R³ and R⁴ in the general formula (I) is a sulfonic group can beobtained, for example, with a sulfur trioxide-trialkylamine complex, asulfur trioxide-pyridine complex, a sulfuric acid-trialkylamine complexor a sulfuric acid-pyridine complex.

In addition, in the general formula (II), R⁵, R⁶, R⁷ and R⁸ eachindependently represent a hydrogen atom or a sulfonic group. Thesulfonic group may have a substituent, as long as the object of thepresent invention is not hindered. A compound in which any of R⁵, R⁶, R⁷and R⁸ in the general formula (II) is a sulfonic group can be obtained,for example, with a sulfur trioxide-trialkylamine complex, a sulfurtrioxide-pyridine complex, a sulfuric acid-trialkylamine complex or asulfuric acid-pyridine complex.

Further, in the above general formula (III), R⁹ and R¹⁰ eachindependently represent a hydrogen atom or a sulfonic group. Thesulfonic group may have a substituent, as long as the object of thepresent invention is not hindered. A compound in which R⁹ and/or R¹⁰ inthe above general formula (III) is a sulfonic group can be obtained, forexample, with a sulfur trioxide-trialkylamine complex, a sulfurtrioxide-pyridine complex, a sulfuric acid-trialkylamine complex or asulfuric acid-pyridine complex.

In addition, in the general formula (IV), R¹¹ and R¹² each independentlyrepresent a hydrogen atom or a sulfonic group. The sulfonic group mayhave a substituent, as long as the object of the present invention isnot hindered. A compound in which R¹¹ and/or R¹² is (are) a sulfonicgroup in the general formula (IV) can be obtained, for example, by asulfur trioxide-trialkylamine complex, a sulfur trioxide-pyridinecomplex, a sulfuric acid-trialkylamine complex or a sulfuricacid-pyridine complex.

Concretely, the saccharide compound of the present invention includes,GlcAβ1-3GalNAc(4,6-O-disulfate)β1-4GlcAβ1-3GalNAc(4,6-O-sulfate),IdoAα1-3GlNAc(4,6-O-disulfate)β1-4IdoAα1-3GalNAc(4,6-O-disulfate),GlcAβ1-3GalNAc(4,6-O-disulfate)β1-4GlcAβ1-3GalNAc(4,6-O-sulfate)β1-4GlcAβ1-3GalNAc(4,6-O-sulfate),IdoAα1-3GlNAc(4,6-O-disulfate)β1-4IdoAα1-3GalNAc(4,6-O-disulfate)β1-4IdoAα1-3GalNAc(4,6-O-disulfate),GlcAβ1-3GalNAc(4,6-O-disulfate)β1-4GlcAβ1-3GalNAc(4,6-O-sulfate)β1-4GlcAβ1-3GalNAc(4,6-O-sulfate)β1-4GlcAβ1-3GalNAc(4,6-O-sulfate),IdoAα1-3GlNAc(4,6-O-disulfate)β1-4IdoAα1-3GalNAc(4,6-O-disulfate)β1-4IdoAα1-3GalNAc(4,6-O-disulfate)β1-4IdoAα1-3GalNAc(4,6-O-disulfate),and the like. Among the above-mentioned saccharide compounds, a compoundhaving a high degree of sulfation is desirable from the viewpoint ofsufficient exhibition of the interactions with L-selectin, P-selectinand chemokine.

The saccharide compound of the present invention can be obtained byperforming the steps of:

(a) digesting a persulfated chondroitin sulfate/dermatan sulfate chainfound in a squid cartilage [Suzuki, S. et al., J. Biol. Chem., 243,1543-1550 (1968)], a mast cell [Katz, H. R. et al., J. Biol. Chem., 261,13393-13396 (1986); and Stevens, R. L. et al., Proc. Natl. Acad. Sci.U.S.A., 85, 2284-2287 (1988)], neutrophil [Ohhashi, Y. et al., Biochem.J., 217, 199-207 (1984); and Petersen. F. et al., J. Biol. Chem., 274,12376-12382 (1999)], monocyte [Uhlin-Hansen, L. et al., J. Biol. Chem.,264, 14916-14922 (1989); McGee, M. P. et al., J. Biol. Chem., 270,26109-26115 (1995)], glomerulus [Kobayashi, S. et al., Biochim. Biophy.Acta, 841, 71-80 (1985)], and glomerulus stromal cell [Yaoita, E. etal., J. Biol. Chem., 265, 522-531 (1990)], with, for example,hyaluronidase, and

(b) subjecting the digestion product obtained in the step (a) to highperformance liquid chromatography, to thereby obtain an oligosaccharidefraction.

In the step (a), a persulfated chondroitin sulfate/dermatan sulfatechain can be obtained by a conventional procedure. In addition, a sourceof the persulfated chondroitin sulfate/dermatan sulfate chain may be,but not particularly limited to the above-exemples, other organisms,tissues, and cells, which carry a persulfated chondroitinsulfate/dermatan sulfate chain.

Then, in the step (b), the digestion product obtained in the above (a)is subjected to chromatography, for example, high performance liquidchromatography. In the step (b), various conventional chromatographiesmay be performed so as to obtain a single kind of oligosaccharide as asingle peak. For example, in the case of squid cartilage-derivedchondroitin A, squid cartilage-derived chondroitin C and squidcartilage-derived chondroitin E, a combination of amine-coupled silicagel column chromatography using a linear gradient of 16 mM to 1MNaH₂PO₄, and subsequent gel filtration column chromatography can beperformed as the above-described chromatography. Here, a solution usedin chromatography may be an aqueous solution or a mixed solution of awater-soluble organic solvent and water, which is suitable forseparating the oligosaccharide and the like.

The saccharide compound obtained in the above step (b) may be sulfatedwith a sulfur trioxide-trialkylamine complex, a sulfur trioxide-pyridinecomplex, a sulfuric acid-trialkylamine complex or a sulfuricacid-pyridine complex. In particular, it is desirable to perform6-O-sulfation of chondroitin sulfate, or 6-O-sulfation of dermatansulfate, preferably, by the method of Nagasawa [Nagasawa, K. et al.,Carbohydr. Res., 158, 183-190 (1986); all teachings of which are herebyincorporated by reference].

The saccharide compound of the present invention exhibits theproperties:

{circle around (1)} interacting, for example, binding with any ofL-selectin, P-selectin and chemokine;

{circle around (2)} regulating, specifically, inhibiting the bindingbetween versican and any of L-selectin, P-selectin and chemokine;

{circle around (3)} inhibiting physiological activities of chemokine.

For this reason, it is expected that the saccharide compound of thepresent invention is applied to treatment or prevention of a disease ofwhich sideration is associated with biological events mediated with anyof L-selectin, P-selectin and chemokine.

Therefore, according to the present invention, there is provided apharmaceutical composition comprising the saccharide compound in thepresent invention as an active ingredient. The pharmaceuticalcomposition may further comprise a carrier (pharmaceutically acceptablecarrier) capable of maintaining stably the effective ingredient, apharmaceutically acceptable auxiliary agent, excipient, binder,stabilizer, buffer, solubilizer, and isotonic agent, depending on thedisease to be applied, the status of the disease, and the individual,organ, local site and a tissue to be administered.

A content of the effective ingredient in the pharmaceutical compositionof the present invention can be appropriately set depending on a diseaseto be applied, status of the disease and an individual, an organ, alocal site and a tissue to be administered and can be set, for example,in the same manner as that of the therapeutic or prophylactic agentdescribed later.

Further, the evaluation of the pharmaceutical composition of the presentinvention can be performed in the same manner as that of the therapeuticor prophylactic agent described later.

Further, according to the present invention, there is provided an agentfor treating or preventing a disease of which sideration is associatedwith biological events mediated by any of L-selectin, P-selectin andchemokine.

One of the features of the therapeutic or prophylactic agent of thepresent invention resides in that the agent comprises the saccharidecompound of the present invention as an active ingredient.

The “biological events mediated by any of L-selectin, P-selectin andchemokine” includes, for example, the infiltration in tissue ininflammation, the regulation of cytokine production, lymphocyte homing,platelet aggregation, vascularization of tumor lesion, cancermetastasis, myocardial ischemia reperfusion disease and the like. Inaddition, the above disease includes inflammatory disease, infectiousdisease, asthma, allergic inflammation, stromal pneumonia, systemicinflammatory response syndrome, and inflammatory disease and the like.

According to the therapeutic or prophylactic agent of the presentinvention, since the agent comprises the saccharide compound of thepresent invention as an active ingredient, there are exhibited excellenteffects such that the therapeutic or prophylactic agent interacts witheach of L-selectin, P-selectin and chemokine, to regulate, specifically,to inhibit eliciting inflammation in inflammatory disease, for example,adhesion of leukocyte with vascular endothelial cell mediated by theL-selectin and/or P-selectin, and to regulate, specifically, to inhibitphysiological activities of chemokine (the regulation of cytokineproduction, Ca²⁺ mobilization and the like). In addition, according tothe therapeutic or prophylactic agent of the present invention, sincethe therapeutic or prophylactic agent comprises the saccharide compoundof the present invention as an active ingredient, there can be regulatedthe binding between at least one kind selected from the group consistingof L-selectin, P-selectin and chemokine, and the ligand thereof.Therefore, according to the therapeutic or prophylactic agent, there areexhibited excellent effects such that there can be carried out treatmentof inflammatory disease, allergic disease, and cancer metastasisassociated with biological events mediated by any of L-selectin,P-selectin and chemokine, or inhibition or prevention of the symptomthereof. Namely, the therapeutic or prophylactic agent of the presentinvention can be used as an anti-inflammatory agent, an antiallergicagent, an anti-cancer agent and the like. Further, according to thetherapeutic or prophylactic agent of the present invention, there areexhibited excellent properties such that the agent improves symptom of adisease such as inflammatory disease, allergic disease, cancermetastasis, myocardial dysfunction, and multiple organ failure, andshows high affinity in a living body. Therefore, there is exhibited anexcellent effect such that the agent can improve symptom of a disease ofwhich sideration is associated with biological events mediated by any ofL-selectin, P-selectin and chemokine.

Evaluation of the therapeutic or prophylactic agent of the presentinvention can be performed, for example, as follows:

Action of the therapeutic or prophylactic agent of the presentinvention, for example, an ability to inhibit the binding, can beevaluated, for example, by surface plasmon resonance analysis describedbelow. Namely, the action of the therapeutic or prophylactic agent ofthe present invention can be evaluated by performing an evaluationmethod comprising the steps of:

(i) immobilizing L-selectin, P-selectin or chemokine on a sensor chip,to obtain each of a L-selectin-immobilized sensor chip, aP-selectin-immobilized sensor chip and a chemokine-immobilized sensorchip,

(ii) loading a ligand for each of L-selectin, P-selectin and chemokineon any of a L-selectin-immobilized sensor chip, a P-selectin-immobilizedsensor chip and a chemokine-immobilized sensor chip, corresponding tothe ligand, at a constant flow rate in the presence of the therapeuticor prophylactic agent of the present invention, and

(iii) detecting the interaction as an optical variation or a massvariation by an appropriate detecting means [e.g. optical detection(fluorescence, fluorescent polarization degree and the like), incombination with mass spectrometer (matrix-assisted laser desorptionionization time of flight mass spectrometer: MALDI-TOF MS,electrospray-ionization mass spectrometer: ESI-MS), thereby obtaining asensorgram.

In the step (i), a ligand may be immobilized on a sensor chip and, inthis case, in the step (ii), any of L-selectin, P-selectin and chemokinemay be loaded on a corresponding ligand-immobilized sensor chip at aconstant flow rate in the presence of the therapeutic or prophylacticagent.

In the evaluation method, in the case where a sensorgram indicating theformation of a complex of each of L-selectin, P-selectin and chemokinewith the ligand thereof in the absence of the therapeutic orprophylactic agent of the present invention is shown, for example, thecase where an optical sensorgram or a mass sensorgram is varied byintroduction of a ligand by liquid pumping, serves as a negativecontrol. Therefore, inhibition of binding by the therapeutic orprophylactic agent of the present invention is recognized as an index,the case where a sensorgram indicating the formation of a complex is notshown or the case where a time until the formation of a complex isdelayed, in the presence of the therapeutic or prophylactic agent of thepresent invention in the same reaction system as in the case of theabsence of the therapeutic or prophylactic agent of the presentinvention.

The action of the therapeutic or prophylactic agent of the presentinvention, for example, the regulation of biological events mediated byany of L-selectin, P-selectin, and chemokine can be evaluated bydetermining the presence or the absence, or an extent of biologicalevents in a cultured cell in the presence of the therapeutic orprophylactic agent of the present invention. For example, regarding Ca²⁺mobilization mediated with secondary lymphoid tissue chemokine, theaction can be evaluated by performing an evaluation method comprisingthe steps of:

i) applying Fura-2 to stimulate to L1.2/CCR7 cell (1×10⁶ cells/ml) withsecondary lymphoid tissue chemokine in the presence or absence of thetherapeutic or prophylactic agent (100 μg/ml) of the present invention,and

ii) measuring a fluorescent rate to monitor an intracellular calciumconcentration according to the description of Hirose, J. et al. [J.Biol. Chem., 276, 5228-5234 (2001); all teachings of which are herebyincorporated by reference].

Further, the action of the therapeutic or prophylactic agent of thepresent invention, for example, a therapeutic or preventive effects onan inflammatory disease in a living body can be evaluated byadministering the therapeutic or prophylactic agent of the presentinvention to an inflammatory disease model animal, and then observing achange in symptom at an inflammatory site; by detecting neutrophilinfiltration using activity of tissue myeloperoxidase as an index.

A dosage form of the therapeutic or prophylactic agent of the presentinvention can be appropriately selected depending on the administrationform. The dosage form includes, for example, a medicine for oraladministration such as a tablet; a medicine for external applicationsuch as a spray medicine and an ointment; injection for subcutaneous,intradermal intramuscular, or intravenous injection, and the like.

Therefore, the content of an effective ingredient in the therapeutic orprophylactic agent of the present invention can be appropriately setdepending on, for example, an age, a weight, pathology and the like ofan individual in need of treatment or prevention of the above disease.It is desired that, for example, the content is 10 to 500 mg, when theadministration form is intravenous injection and that the content is 10to 500 mg, when the administration form is subcutaneous injection.

In addition, a dose of the therapeutic or prophylactic agent of thepresent invention to an individual can be appropriately set dependingon, for example, the age, weight, pathology and the like of anindividual in need of treatment or prevention of the above disease. Itis desired that, for example, the amount of an effective ingredient is 1μg/kg (weight of individual) to 10 mg/kg (weight of individual),preferably 100 μg/kg (weight of individual) to 10 mg/kg (weight ofindividual), more preferably 1 mg/kg (weight of individual) to 8 mg/kg(weight of individual).

The therapeutic or prophylactic agent of the present invention mayfurther comprise a pharmaceutically acceptable auxiliary agent,excepient, binder, stabilizer, buffer, solubilizer and isotonic,depending on status of a disease of which sideration is associated withbiological events mediated by any of L-selectin, P-selectin andchemokine, and the individual, organ, local site and tissue to beadministered and the like.

In addition, the therapeutic or prophylactic agent of the presentinvention may be an agent obtained by making a carrier (pharmaceuticallyacceptable carrier) capable of stably maintaining the effectiveingredient to carry the effective ingredient. Specifically, for example,the effective ingredient may be carried by a pharmaceutically acceptablecarrier which can facilitate introduction into a living body such as anindividual, organ, local site and tissue to be administered.

Further, according to the present invention, there is provided a methodfor treating or preventing a disease associated with biological eventsmediated by any of L-selectin, P-selectin and chemokine, specifically,for example, a method for treating inflammatory disease, allergicdisease, cancer metastasis, myocardial dysfunction, multiple organfailure and the like, inhibiting or preventing symptom thereof. Themethod for treatment or prevention of the present invention can beperformed according to a dosage form and a dose of the above therapeuticor prophylactic agent.

The present invention will be explained in more detail below byExamples, but the present invention is not limited by the Examples.Unless otherwise is indicated, reagents used in the present Examples arethe same as those described in Kawashima, H. et al. [J. Biol. Chem.,275, 35448-35456 (2000)] and Hirose, J. et al. [J. Biol. Chem., 276,5228-5234 (2001)]. In addition, for a composition of a cell culturemedium etc., see Frshney, R. Ian, Culture of animal cell: A manual ofbasic technique, 2nd ed., Alan R. Liss. Inc., 66-84 (1987), allteachings of which are hereby incorporated by reference.

EXAMPLE 1

Requirement of sulfation in the interaction between a chondroitinsulfate/dermatan sulfate chain (hereinafter, also referred to as CS/DSchain) in versican, and any of L-selectin, P-selectin and CD44 wasexamined by treating ACHN cells with a metabolism inhibitor forsulfation, sodium chlorate.

(1) Preparation of Human CD44-Ig

Human-derived CD44 cDNA was prepared by PCR using a sense primer (SEQ IDNO: 1): 5′-TTTAAGCTT ATGGACAAGTTTTGGTGGCAC-3′ (SEQ ID NO: 1)wherein the bold face is HindIII restriction enzyme recognition site,and the underlined portion is codons for initial 7 amino acids of humanCD44,

and an antisense primer (SEQ ID No.: 2): (SEQ ID NO: 2)5′-TTTTCTAGAAACACGTCATCATCAGTAGGGTT-3′wherein the bold face is XbaI restriction enzyme recognition site, andthe underlined portion is codons of amino acids positions 172 to 178 ofhuman CD44.The resulting amplification product was inserted into a cloning site ofthe expression vector pcDNA 3.1/Myc-His(+) B [manufactured byInvitrogen], to obtain a human CD44 expression vector. In addition, theamplification product was sequenced.

293T cells were transfected with the human CD44 expression vector, usingthe LipofectAMINE™ reagent [manufactured by Invitrogen] according to theinstruction by the manufacturer. The resulting transfected cells werecultured at 37° C. for 4 days in the Dulbecco's modified Eagle mediumcontaining 20% by weight of bovine fetal serum in a CO₂ incubator, toobtain a conditioned medium.

Thereafter, the conditioned medium was subjected to immunoaffinitychromatography with anti-CD44 monoclonal antibody BRIC 235-coupledCNBr-activated Sepharose 4B, to obtain monomeric CD44. The resultingsoluble CD44 was subjected to SDS-PAGE and silver staining. As a result,the purity of the resulting soluble CD44 was more than 95%.

Then, using the resulting CD44, human CD44-immunoglobulin (Ig) wasprepared according to the method described in Toyama-Sorimachi, N. [J.Biol. Chem., 270, 7437-7444, (1995); all teachings of which are herebyincorporated by reference].

(2) Labeling of Metabolism of ACHN Cells Using [³⁵S]Sodium Sulfate or[³⁵S]Methionine.

ACHN cells were pre-incubated at 37° C. for 6 hours in RPMI 1640containing 10% by weight of bovine fetal serum in the presence orabsence of 30 mM sodium chlorate. A confluent monolayer of the resultingACHN cells was labeled by incubation for 18 hours in the presence orabsence of 30 mM sodium chlorate, in a medium of either of:

(i) Eagle minimum basal medium SMEM [sulfate-free; manufactured byBioWhittaker] containing 2% by weight of dialyzed bovine fetal serum inthe presence of 0.2 mCi/ml [³⁵S]Na₂SO₄ [trade name: Sulfer-35;manufactured by ICN Radiochemicals], or

(ii) methionine-free RPMI 1640 medium [manufactured by Invitrogen]containing 2% by weight of dialyzed bovine fetal serum in the presenceof 0.2 mCi/ml [³⁵S]-methionine [manufactured by ICN Radiochemicals].

(3) Immunoprecipitation

Immunoprecipitation was performed as follows, according to the methoddescribed in the publication of Kawashima, H. et al. [Int. Immunol., 11,393-405 (1999); all teachings of which are hereby incorporated byreference] except that beads were washed using buffer A (0.05% Tween 20,20 mM HEPES-NaOH, 0.15M NaCl, 1 mM CaCl₂, 1 mM MgCl₂, pH6.8).

Each of the antibody and the Ig chimera shown in FIG. 1 (equivalent to10 μg) was coupled to Protein A-Sepharose column (equivalent to 10 μL)by a conventional method, to obtain antibody-coupled Protein A-Sepharoseor Ig chimera-coupled Protein A-Sepharose. Then, each antibody-coupledProtein A-Sepharose or each Ig chimera-coupled Protein A-Sepharose(equivalent to 10 μl gel), and the conditioned medium were incubated at4° C. overnight in 1 ml of the buffer A. Anti-D [Zimmermann, D. R. etal., J. Cell. Biol., 124, 817-825 (1994)] which is an anti-versicanpolyclonal antibody was provided by Dr. Dietel R. Zimmermann (Instituteof Clinical Pathology, University of Zuerich), and human L-selectin-Igwas manufactured by R & D Systems Inc.

Thereafter, the beads were washed with the buffer A, to obtainprecipitates. The resulting precipitates were subjected toSDS-agarose-PAGE, and thereafter, signal ascribed to ³⁵S label on a gelwas detected by autoradiography. The results are shown in FIG. 1.

As shown in lanes 5 to 8 of FIG. 1, although a core protein of versicanwas synthesized, as shown in lanes 1 to 4, treatment with sodiumchlorate resulted in more than 90% inhibition of sulfation relative tothe case without treatment.

In addition, as shown in lanes 11, 12, 15 and 16 of FIG. 1, the abovesodium chlorate treatment inhibited the interaction between versican andeach of L-selectin-Ig and P-selectin-Ig. Therefore, it was suggestedthat sulfation of CS/DS chain of versican is necessary for theinteraction between versican and each of L-selectin and P-selectin. Onthe other hand, as shown in lanes 13 and 14 of FIG. 1, unsulfatedversican did not interact with E-selectin-Ig, which is consistent withthe results in Kawashima, H. et al. [J. Biol. Chem., 275, 35448-35456,(2000)].

In contrast, as shown in lanes 17 and 18 of FIG. 1, the interactionbetween versican and CD44-Ig was not inhibited by sodium chloratetreatment. However, the fact that the interaction between versican andCD44-Ig was not inhibited by sodium chlorate treatment was contemplatedto be due to the formation of a trimolecular complex composed ofversican, hyaluronic acid and CD44-Ig.

Then, [³⁵S]methionine-labeled conditioned medium of ACHN cells wastreated with hyaluronidase SD (in FIG. 1, “HA′ase”; 50 mU/ml) for 3hours in the same manner as described in the publication [Kawashima, H.et al., J. Biol. Chem., 275, 35448-35456, (2000); all teachings of whichare hereby incorporated by reference], and then the resultingconditioned medium and CD44-Ig were incubated.

As the result, although the hyaluronic acid was almost completelydegraded and removed by the above treatment with hyaluronidase SD, butas shown in lanes 19 and 20 of FIG. 1, the interaction between versicanand CD44-Ig was not affected at all. Therefore, it was confirmed thatsulfation of versican is not necessary for the interaction of versicanand CD44, namely, a CS/DS chain of versican interacts with CD44 in asulfation independent manner.

EXAMPLE 2

The structure for interacting with each of selectin and CD44 wasexamined by performing Inhibition assay using various CS/DS chains.

(1) Analysis of Disaccharide Composition of CS/DS Chain

A CS/DS chain used in the inhibition assay was chondroitin (“CH” inTable 1), chondroitin sulfate A (“CS A” in Table 1), chondroitinpolysulfate (“CPS” in Table 1), dermatan, dermatan sulfate purified froma cock's comb [“DS” in Table 1; manufactured by SEIKAGAKU CORPORATION;Nagasawa, K. et. al., Carbohydr. Res., 131, 301-314, (1984)], dermatanpolysulfate (“DPS” in Table 1), and chondroitin sulfate E (“CS E” inTable 1). The above chondroitin was produced by chemical desulfurizationof chondroitin sulfate A derived from whale cartilage [Nagasawa, K., J.Biochem., 86, 1323-1329 (1979)]. The above chondroitin polysulfate(manufactured by SEIKAGAKU CORPORATION) was produced by selective6-O-sulfation of chondroitin sulfate A [Nagasawa, K. et al., Carbohydr.Res., 158, 183-190 (1986)]. The above dermatan polysulfate (manufacturedby SEIKAGAKU CORPORATION) was produced by selective 6-O-sulfation ofdermatan sulfate [Nagasawa, K. et al., Carbohydr. Res., 158, 183-190(1986)]. Dermatan (manufactured by SEIKAGAKU CORPORATION) was producedby the above chemical desulfurization of dermatan sulfate [Nagasawa, K.,J. Biochem., 86, 1323-1329 (1979)].

The CS/DS chain was treated with chondroitinase ABC (1 unit/ml) at 37°C. for 7 hours in the same manner as that of Fujimoto, T. et al. [Int.Immunol., 13, 359-366 (2001); all teachings of which are herebyincorporated by reference]. The resulting product was subjected to HPLCwith amine-coupled silica PA-03 column, to thereby determine thedisaccharide composition. Results are shown in Table 1. TABLE 1 ΔDi-ΔDi- ΔDi- ΔDi-di ΔDi-di ΔDi-di ΔDi-tri 0S 6S 4S (2,6)S (2,4)S (4,6)S(2,4,6)S CH 94.7 2 3.3 CS A 2.2 23.7 74.1 CPS 26.9 3.3 10.9 47.3 11.5Dermatan 85.8 14.2 DS 5 3.4 85.2 6.3 DPS 1.8 9.1 1.5 2.9 62.1 21.2 CS E5.8 9.7 18.7 65.9

Although a chain length and an atomic valence of chondroitinpolysulfate, those of chondroitin sulfate A and those of chondroitin areidentical, degrees of sulfation of these glycosaminoglycans aredifferent. Similarly, dermatan polysulfate, dermatan sulfate anddermatan are different only in sulfation.

(2) Binding Inhibition Assay

Inhibition assay of binding between versican and each of L-selectin,P-selectin and CD44 was performed as follows. First, each well on a 96well flat bottom microtiter plate (Coster EIA/RIA plate No. 3690) wascoated with L-selectin-Ig (3 μg/ml), P-selectin-Ig (4 μg/ml) or CD44-Ig(0.25 μg/ml) by a conventional procedure. Then, various concentrationsof each of keratan sulfate, chondroitin, chondroitin sulfate A,chondroitin polysulfate, dermatan, dermatan sulfate, dermatanpolysulfate and chondroitin sulfate E, and biotinylated versican wasadded to the wells on the plate. The plate was incubated at roomtemperature for 2 hours. Thereafter, the plate was washed with thebuffer A, and enzyme-linked immunosorbent assay was performed in thesame manner as described in Kawashima, H. et al. [J. Biol. Chem., 275,35448-35456 (2000)]. Binding was determined by measuring absorbance at620 nm using alkaline phosphatase-conjugated streptavidin and Blue Phos™substrate.

Results are shown in FIG. 2. In FIG. 2, a cross denotes keratan sulfate,an open triangle denotes chondroitin, an open square denotes chondroitinsulfate A, an open circle denotes chondroitin polysulfate, a solidtriangle denotes dermatan, a solid square denotes dermatan sulfate, asolid circle-solid line denotes dermatan polysulfate, and a solidcircle-dashed line denotes chondroitin sulfate E. In FIG. 2, panel A isthe results for binding between versican and L-selectin, panel B beingthe results regarding binding between versican and P-selectin, and panelC being the results regarding binding between versican and CD44.

As shown in FIG. 2, binding between biotinylated versican and each ofL-selectin-Ig and P-selectin-Ig is inhibited by glycosaminoglycancontaining GlcAβ1/IdoAα1-3GalNAc(4,6-O-disulfate) as a main disaccharidecomponent (chondroitin polysulfate, dermatan polysulfate and chondroitinsulfate E) in a dose-dependent manner, but was not inhibited by alow-sulfated or unsulfated CS/DS chain such as chondroitin, chondroitinsulfate A, dermatan, and dermatan sulfate, or keratan sulfate. Incontrast, binding between biotinylated versican and CD44-Ig wasinhibited by all of CS/DS chains examined such as low-sulfated CS/DSchain, and unsulfated CS/DS chain, but was not inhibited by keratansulfate.

These results were consistent with the results of the above Example 1that sulfation plays an important role in the interaction between CS/DSchain of versican and each of L-selectin and P-selectin, but does notplay a role in the interaction between a CD/DS chain of versican andCD44.

EXAMPLE 3

The glycosaminoglycan structure of versican was characterized.

(1) Preparation of Versican-Derived Glycosaminoglycan

Purified versican (80 μg) was incubated at 37° C. for 48 hours in 2 mlof a solution (composition: 5 mM Tris-HCl, 5 mM CH₃COONa, 1 mM CaCl₂, 1mM MgCl₂, pH8.0) containing pronase [90 U or more, manufactured byCalbiochem]. After incubation, 0.5 ml of a solution (composition: 1MNaBH₄, 5M NaOH) was added to the resulting product, followed byincubation at 37° C. After incubation for 24 hours, 0.5 ml of CH₃COOHwas added to the resulting product to terminate the reaction, to therebyobtain the reaction mixture containing versican-derivedglycosaminoglycan. The resulting reaction mixture was dialyzed againstdistilled water using a Spectra/Por dialysis membrane [molecular weightexclusion limit 3,500; manufactured by The Spectrum Co.], to obtainversican-derived glycosaminoglycan.

(2) High Sensitive Disaccharide Composition Analysis of Versican-DerivedGlycosaminoglycan

The versican-derived glycosaminoglycan obtained in the above item (1)and chondroitinase ABC (0.38 U/ml) were incubated at 37° C. for 1 hourin the presence or the absence of chondro-6-sulfatase [0.3]U/ml buffer B(3% acetic acid adjusted to pH 7.0 with triethylamine)], and thereafter,the resulting product was dried to obtain a disaccharide product. Then,the disaccharide product (0.1 to 50 nmol) and 5 μl of a derivatizationreagent mixture [composition: 0.35M 2-aminobenzoamide (2-AB), 0.1MNaCNBH₄, 30% by weight acetic acid in dimethyl sulfoxide] were mixed andthen incubated at 65° C. for 2 hours according to the method ofKinoshita et al. [Kinoshita A. et al., Anal. Biochem., 269, 367-378(1999)]. Then, the resulting product was fractionated withchloroform:distilled water (1:1), to collect the aqueous layercontaining a derivatized disaccharide.

The resulting aqueous layer containing a derivatized disaccharide wasanalyzed by high performance liquid chromatography (HPLC) as describedin that of the previous report [Fujimoto, T. et al., Int. Imminol., 13,359-366 (2001)]. Regarding the extract, each of the excision wavelengthat 330 nm and the emission wavelength at 420 nm was monitored using afluorescent detector. The results are shown in FIG. 3. In FIG. 3, 0Sdenotes an elution position of 2-AB-derivatized ΔDi-0S, 4S denotes anelution position of 2-AB-derivatized ΔDi-4S, 6S denotes an elutionposition of 2-AB-derivatized ΔDi-6S, diS_(D) denotes an elution positionof 2-AB-derivatized ΔDi-di(2,6)S, diS_(E) denotes an elution position of2-AB-derivatized ΔDi-di(4,6)S, and UA2S denotes an elution position of2-AB-derivatized ΔDi-UA2S. In addition, a table of abbreviations ofdisaccharide specimens is shown in Table 2. TABLE 2 Abbreviation SugarChain ΔDi-0S Δ^(4,5)HexAα1-3GalNAc ΔDi-4SΔ^(4,5)HexAα1-3GalNAc(4-O-sulfate) ΔDi-6SΔ^(4,5)HexAα1-3GalNAc(6-O-sulfate) ΔDi-di(2,6)SΔ^(4,5)HexA(2-O-sulfate)α1-3GalNAc(6-O-sulfate) ΔDi-di(2,4)SΔ^(4,5)HexA(2-O-sulfate)α1-3GalNAc(4-sulfate) ΔDi-di(4,6)SΔ^(4,5)HexAα1-3GalNAc(4,6-O-disulfate) ΔDi-tri(2,4,6)SΔ^(4,5)HexA(2-O-sulfate)α1-3GalNAc(4,6-O-disulfate) ΔDi-UA2SΔ^(4,5)HexA(2-O-sulfate)α1-3GalNAc Di-0S GlcAβ1-3 GalNAc Di-4SGlcAβ1-3GalNAc(4-O-sulfate) Di-6S GlcAβ1-3GalNAc(6-O-sulfate)Di-di(4,6)S GlcAβ1-3GalNAc(4,6-O-disulfate)

As shown in panel A in FIG. 3, five peaks ascribed to elution positionsof disaccharide specimens were detected for versican-derivedglycosaminoglycan treated with chondroitinase ABC. In addition, as shownin panel B of FIG. 3, three peaks ascribed to ΔDi-0S, ΔDi-UA-2S andΔDi-4S were detected for versican-derived glycosaminoglycan treated withchondro-6-sulfatase and chondroitinase ABC. Therefore, it was confirmedthat the five peaks detected in panel A of FIG. 3 are ΔDi-0S, ΔDi-6S,ΔDi-4S, ΔDi-di(2,6)S and ΔDi-di(4,6)S.

The peak area of each ΔDi-0S, ΔDi-6S, ΔDi-4S, ΔDi-di(2,6)S andΔDi-di(4,6)S in panel A of FIG. 3 was 0.8%, 15.7%, 77.6%, 1.4% and 4.5%.Although a small additional peak (6.3%) not ascribed to a disaccharidespecimen was detected, the similar results was also obtained usingchondroitinase ACII in place of chondroitinase ABC. From these results,it was suggested that glycosaminoglycan of versican containsGlcAβ1-3GalNAc(4,6-O-disulfate), and is a heteropolymer composed of amixture of a major CS chain, and a minor DS chain which is resistant tochondroitinase ACII.

EXAMPLE 4

It is shown that versican can interact not only with an adhesionmolecule but also with a certain chemokine [Hirose, J. et al., J. Biol.Chem., 276, 5228-5234 (2001)]. Then, the requirement of sulfation forthe interaction between versican and chemokine was examined.

(1) Preparation of Low-Sulfated Versican

ACHN cells were cultured for 2 days in the presence or absence of 100 mMsodium chlorate in RPMI 1640 containing 10% by weight of bovine fetalserum. After the conditioned medium was removed, cells were furthercultured for 4 days in a serum-free medium, EX-CELL 610 HSF[manufactured by JRH Bioscience] in the presence or absence of 100 mMsodium chlorate. The conditioned medium was recovered, and spun at10,000×g at 4° C. for 15 minutes to obtain each of a sodiumchlorate-treated conditioned medium and sodium chlorate-untreatedconditioned medium.

The sodium chlorate-treated conditioned medium or the sodium chlorateuntreated conditioned medium, and 20 turbidity reduction unit/mlhyaluronidase (Streptomyces hyalurolyticus) were incubated at 37° C. for4.5 hours. Thereafter, each of the resulting products, and anti-Dantibody (10 μg)-coupled Protein A-Sepharose beads (10 μl beads) wasincubated, to precipitate versican from each conditioned medium. Theresulting beads were washed, and incubated at 37° C. for 2 hours in thepresence of 1 unit/ml chondroitinase ABC and 1 unit/ml chondroitinaseACII. Thereafter, the disaccharide product was recovered, andderivatized with 2-AB according to the above method of Kinoshita et al.The resulting derivatized disaccharide product was analyzed by HPLC. Theresults are shown in panel A of FIG. 4. In panel A of FIG. 4, 0S denotesan elution position of 2-AB-derivatized ΔDi-0S, 4S being an elutionposition of 2-AB-derivatized ΔDi-4S, 6S being an elution position of2-AB-derivatized ΔDi-6S, diS_(D) being an elution position of2-AB-derivatized ΔDi-di(2,6)S, and diS_(E) being an elution position of2-AB-derivatized ΔDi-di(4,6)S.

As shown in panel A of FIG. 4, in the case of versican-derivedglycosaminoglycan obtained from the sodium chlorate-treated conditionedmedium, only ΔDi-0S was detected as a main peak (82.8%) inversican-derived glycosaminoglycan, ΔDi-6S (7.2%) and ΔDi-4S (10.0%)were detected as a minor peak, and ΔDi-di(2,6)S or ΔDi-di(4,6) was notdetected. Therefore, it is found that sodium chlorate treatment providesmainly low-sulfated versican capable of generating a unsulfated CS/DSchain.

(2) Sandwich-Type Enzyme-Linked Immunosorbent Assay

Binding between each of the resulting low-sulfated versican and intactversican, and chemokine was examined by sandwich-type enzyme-linkedimmunosorbent assay.

A well of a 96 well flat bottom microtiter plate was coated with BSA (6μg/ml), anti-versican monoclonal antibody 2B1 (“2B1” in FIG. 4; 5μg/ml), L-selectin-Ig (“L-Ig” in FIG. 4; 3 μg/ml), E-selectin-Ig (“E-Ig”in FIG. 4; 3 μg/ml), P-selectin-Ig (“P-Ig” in FIG. 4; 3 μg/ml), CD44-Ig(3 μg/ml), secondary lymphoid tissue chemokine (“SLC” in FIG. 4; 3μg/ml), C-terminal truncated secondary lymphoid tissue chemokine(“SLC-T” in FIG. 4; 3 μg/ml), γ-interferon inducible protein-10 (“IP-10”in FIG. 4; 3 μg/ml), platelet factor 4 (“PF4” in FIG. 4; 6 μg/ml),stromal cell-derived factor-1β (“SDF-1β” in FIG. 4; 6 μg/ml) or stromalcell-derived factor-1α (“SDF-1α” in FIG. 4; 6 μg/ml), and then blockedwith phosphate buffered physiological saline containing 3% by weight ofBSA. The chemokine provided by Dr. Melissa Swoop Wills [VertexPharmaceutical Co.] was used as the above C-terminal truncated secondarylymphoid tissue chemokine.

A conditioned medium of sodium chlorate-treated ACHN cells or aconditioned medium of sodium chlorate-untreated ACHN cells was added toa well of the resulting plate, followed by incubation for 1 hour. Afterthe well was washed with the buffer A, 1 μl/ml a biotinylated anti-Dantibody was added thereto, followed by incubation for 1 hour. Bindingwas determined by measuring absorbance at 620 nm using alkalinephosphatase-conjugated streptavidin and Blue Phos™ substrate. Theresults are shown in panel B of FIG. 4. In panel B of FIG. 4, a blackbar shows results of the case where versican derived from untreatedconditioned medium was used, and a hatched bar shows results of the casewhere versican derived from sodium chlorate-treated conditioned mediumwas used. Each bar shows mean±standard deviation of tetraplicatemeasurements.

As shown in panel B of FIG. 4, both of intact versican and low-sulfatedversican bound to anti-versican monoclonal antibody 2B1 and CD44-Ig, andonly intact versican bound with L-selectin-Ig and P-selectin-Ig, whichis consistent with the results shown in FIG. 1.

In addition, as shown in panel B of FIG. 4, versican which is intact butis not low-sulfated (black bar) remarkably bound with chemokine such assecondary lymphoid tissue chemokine, γ-interferon inducible protein-10,platelet factor 4, and stromal cell-derived factor-1β. Therefore, it wassuggested that sulfation in a CS/DS chain of versican is necessary forthe interaction with chemokine.

Further, as shown in panel B of FIG. 4, any type of intact andlow-sulfated versicans scarcely bound to recombinant truncated-typesecondary lymphoid tissue chemokine lacking C-terminal 32 amino acidscontaining a basic amino acid cluster or, if any, slightly boundthereto. In addition, any type of intact and low-sulfated versicans didnot bind to stromal cell-derived factor-1α naturally defective inC-terminal 4 amino acids of stromal cell-derived factor-1β. From theseresults, it was suggested that a CS/DS chain of versican interacts witheach of a C-terminal region of secondary lymphoid tissue chemokine and aC-terminal region of stromal cell-derived factor-1β.

EXAMPLE 5

Effects of a persulfated CS/DS chain on binding between versican andchemokine was examined.

A well of a 96 well flat bottom microtiter plate was coated with BSA (5μg/ml), anti-versican monoclonal antibody 2B1 (5 μg/ml), CD44-Ig (7μg/ml), L-selectin-Ig (3 μg/ml), P-selectin-Ig (3 μg/ml) or chemokine (1μg/ml), and then blocked with a phosphate buffered physiological salinecontaining 3% by weight of BSA. As the above chemokine, secondarylymphoid tissue chemokine, γ-interferon inducible protein-10, plateletfactor 4, stromal cell-derived factor-1β [manufactured by Pepro Tech]and stromal cell-derived factor-1α [manufactured by Pepro Tech] wereused.

In the well on the resulting plate, biotinylated versican (0.25 μg/ml)was incubated at room temperature for 2 hours in the presence or absenceof 100 μg/ml of each glycosaminoglycan (chondroitin, chondroitin sulfateA, chondroitin polysulfate, dermatan polysulfate and chondroitin sulfateE). Thereafter, the plate was washed with the buffer A, andenzyme-linked immunosorbent assay was performed in the same manner asthat of the previous report [Kawashima, H. et al., J. Biol. Chem., 275,35448-35456 (2000)]. Binding was determined by measuring absorbance at620 nm using alkaline phosphatase-conjugated streptavidin and Blue Phos™substrate. Results are shown in FIG. 5. In FIG. 5, expression of anabscissa axis is the same as that of FIG. 4. In each lane, bar 1 showsthe results in the absence of glycosaminoglycan, bar 2 beingchondroitin, bar 3 being chondroitin sulfate A, bar 4 being chondroitinpolysulfate, bar 5 being dermatan polysulfate, and bar 6 beingchondroitin sulfate E.

As shown in FIG. 5, each binding between biotinylated versican and eachof secondary lymphoid tissue chemokine, γ-interferon inducibleprotein-10, platelet factor 4, stromal cell-derived factor-1β,L-selectin and P-selectin was inhibited by a persulfated CS/DS chainsuch as chondroitin polysulfate, dermatan polysulfate, and chondroitinsulfate E to the same degree. In addition, as shown above, althoughbinding between biotinylated versican and CD44-Ig was inhibited by eachof chondroitin, chondroitin sulfate A, dermatan polysulfate andchondroitin sulfate E, the effects of chondroitin polysulfate were notremarkable in the dose range used in the present experimental system.Binding between versican and anti-versican monoclonal antibody 2B1 wasnot affected by addition of any of examined glycosaminoglycans. Namely,from these results, it was suggested that sulfation of a CS/DS chain wasnecessary for the interaction with chemokine, and that a persulfatedCS/DS chain containing GlcAβ1/IdoAα1-3GalNAc(4,6-O-disulfate) interactedwith chemokine.

EXAMPLE 6

Affinity kinetics of the interaction between various CS/DS chains andeach of L-selectin, P-selectin, CD44 and chemokine were examined bysurface plasmon resonance using BIAcore™ biosensor [manufactured byBIAcore AB].

All experiments were performed at 25° C. At stages of washing anddissociation, buffer C (20 mM HEPES-NaOH, 0.15M NaCl, 1 mM CaCl₂, 1 mMMgCl₂, pH6.8) was used as a running buffer.

First, about 1.8 to 2.0 kiloresonance unit (1 kiloresonance unit=1ng/mm²) of streptavidin were covalently immobilized onto B1 sensor chipvia a primary amine group using an amine coupling kit [manufactured byAmercham-Bioscience] according to the instruction provided by themanufacturer. The remaining activated groups were blocked with 150 μl of1M ethanolamine-HCl (pH8.5). Then, each glycosaminoglycan which had beenbiotinylated at a reducing end was injected to a sensor tip surface byusing EZ-link™ biotin-LC-hydrazide [manufactured by Pierce] so as toobtain about 150 resonance unit of an immobilization level, according tothe method of Sadir et al. [J. Biol. Chem., 276, 8288-8296 (2001); allteachings of which are hereby incorporated by reference].

Binding assay was performed by injecting continuously each of variousconcentrations of secondary lymphoid tissue chemokine, γ-interferoninducible protein-10, stromal cell-derived factor-1β, monomericL-selectin [manufactured by Genzyme-Techne], monomeric P-selectin[manufactured by Genzyme-Techne] and monomeric CD44, to aglycosaminoglycan-coupled sensor tip at a flow rate of 30 μl/min for 0to 90 seconds, and then injecting a running buffer thereto. A responsein resonance unit was recorded as a function of a time.

A sensor tip surface was regenerated with 300 μl of 1 M NaCl whenchemokine or CD44 was used, or regenerated with 300 μl of 1 M NaCl, andadditional 100 μl of 50 mM EDTA (pH8.0), when selectin was used. As aresult of regeneration of a sensor tip surface, a remarkable change in abaseline was not observed.

An affinity kinetic parameter was determined by BIAevaluation 3.0software [manufactured by Pharmacia Biosensor] using a single sitebinding model. FIG. 6 shows a sensorgram of BIAcore recording theinteraction between immobilized glycosaminoglycan, and each ofchemokine, L-selectin and CD44. Table 3 shows kinetic parameters of theinteraction [association rate constant (k_(on)), dissociation rateconstant (k_(off)) and equilibrium dissociation constant (k_(d))]. InFIG. 6 and Table 3, SLC denotes secondary lymphoid tissue chemokin,IP-10 denotes γ-interferon inducible protein-10, SDF-1β denotes stromalcell-derived factor-1β, CS E denotes chondroitin sulfate E, and CS Adenotes chondroitin sulfate A. In addition, in Table 3, K_(d) value forthe interaction between monomeric CD44 and chondroitin sulfate E is avalue calculated from a binding amount at equilibrium, and K_(d) valueof the interaction between CD44 and hyaluronic acid is a valuecalculated using CD44-Ig. TABLE 3 k_(on) k_(off) K_(d) Protein GAG(M⁻¹s⁻¹) (s⁻¹) (nM) L-selectin CS E 2.07 × 10⁴ 4.39 × 10⁻⁴ 21.2 CPS 7.68× 10³ 3.66 × 10⁻⁴ 47.7 DPS 1.37 × 10⁴ 2.89 × 10⁻⁴ 21.1 P-selectin CS E4.21 × 10⁴ 1.25 × 10⁻³ 29.7 CPS 1.34 × 10⁴ 7.59 × 10⁻⁴ 56.8 DPS 2.40 ×10⁴ 6.64 × 10⁻⁴ 27.7 CD44 CS E ^(a) ND ^(b) ND 2.11 × 10⁵ CH 5.03 × 10²6.49 × 10⁻² 1.29 × 10⁵ CS A 5.76 × 10² 4.90 × 10⁻² 8.52 × 10⁴ HA ^(c)2.61 × 10⁴ 5.43 × 10⁻² 2.08 × 10³ SLC CS E 4.15 × 10⁴ 3.56 × 10⁻³ 85.8CPS 2.42 × 10⁴ 2.44 × 10⁻³ 101 DPS 8.64 × 10³ 2.78 × 10⁻⁴ 32.2 IP-10 CSE 1.62 × 10⁴ 2.11 × 10⁻³ 130 CPS 3.15 × 10⁴ 2.30 × 10⁻³ 73.2 DPS 2.64 ×10⁴ 9.23 × 10⁻⁴ 34.9 SDF-1β CS E 1.81 × 10⁴ 5.30 × 10⁻³ 293 CPS 3.52 ×10⁴ 3.04 × 10⁻³ 86.3^(a) Kd value for the interaction between monomeric CD44 and CS E wascalculated from the amount bound at equilibrium.^(b) ND, not determined.^(c) Kd value for the interaction between CD44 and HA was determinedusing CD44-Ig. Affinity kinetic parameters were determined with the BIAevaluation 3.0 software using the bivalent analyte binding model.

As shown in FIG. 6, secondary lymphoid tissue chemokine, γ-interferoninducible protein-10, stromal cell-derived factor-1β, monomericL-selectin and monomeric P-selectin bound to chondroitin sulfate Eimmobilized on a sensor tip surface in a dose dependent manner. Thesimilar affinity kinetic was observed when chondroitin polysulfate ordermatan polysulfate was used in place of chondroitin sulfate E. Incontrast, selectin and chemokine did not interact with chondroitinsulfate A or chondroitin. These results were consistent with the resultsof the above inhibition assay shown in FIG. 2 and FIG. 5.

Further, evaluation of the affinity kinetic parameters shown in Table 3showed that L-selectin, P-selectin and chemokine interact with apersulfated CS/DS chain containingGlcAβ1/IdoAα1-3GalNAc(4,6-O-disulfate) with high affinity (K_(d): 21.1to 293 nM). In contrast, it was shown that the interaction between CD44and glycosaminoglycan is considerably different from the interactionbetween selectin and glycosaminoglycan, and the interaction betweenchemokine and glycosaminoglycan. In addition, from results of Table 3,it was shown that monomeric CD44 bound to chondroitin sulfate A orchondroitin with low affinity (K_(d): 85.2 to 129 μM). Monomeric CD44weakly bound to chondroitin sulfate E (K_(d): 211 μM), and did not bindto chondroitin polysulfate or dermatan polysulfate. All of examinedCS/DS chains hardly interacted with monomeric E-selectin, C-terminaltruncated secondary lymphoid tissue chemokine and stromal cell-derivedfactor-1α, or if any, slightly interacted therewith. On the other hand,as shown in Table 3, a persulfated CS/DS chain interacted withparticular chemokine with high affinity as in the case of L-selectin orP-selectin. High affinity binding between a persulfated CS/DS chain andchemokine (secondary lymphoid tissue chemokine, γ-interferon inducibleprotein-10, and stromal cell-derived factor-1β) suggested that thesechemokines are immediately trapped by a persulfated CS/DS chain in vivo.This hypothesis is supported by kinetic analysis by surface plasmonresonance analysis showing that the formation of a persulfatedCS/DS-chemokine complex is characterized by a high association rate(0.864 to 4.15×10⁴ M⁻¹s⁻¹).

Previously, it was reported that monomeric L-selectin binds toimmobilized glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1)with low affinity (K_(d)=108 μM) at a very high association rate (≧10⁵M⁻¹s⁻¹) and dissociation rate (≧10⁵ s⁻¹) [Nicholson, M. W. et al., J.Biol. Chem., 273, 763-770 (1998)]. In addition, it was reported thatmonomeric P-selectin binds to P-selectin glycoprotein ligand-1 withrelatively high affinity (K_(d): about 300 nM) at a high associationrate (4.4×10⁶ M⁻¹s⁻¹) and dissociation rate (1.4 s⁻¹) [Mehta, P. et al.,J. Biol. Chem., 273, 32506-32513 (1998)]. It is presumed that theseproperties are important in selectin-mediated rolling adhesion dynamicsmediated by rapid adhesion and deadhesion.

However, surprisingly, as shown in FIG. 6 and Table 3, the surfaceplasmon resonance analysis showed that binding affinity between apersulfated CS/DS chain and each of L-selectin and P-selectin is higherthan binding affinity between the known ligand and each of L-selectinand P-selectin. Therefore, when an appropriate CS/DS chain is locallyexpressed, it is thought that high affinity binding between apersulfated CS/DS chain and each of L-selectin and P-selectin at a lowdissociation rate as shown in Table 3 allows for leukocyte rollinginteraction and/or static adhesion interaction at a different rollingrate.

EXAMPLE 7

Various oligosaccharide fragments were prepared from each of chondroitinsulfate A, chondroitin sulfate C and chondroitin E by digestion withovine testis hyaluronidase, to determine a structural unit whichdirectly interacts with each of L-selectin, P-selectin, CD44 andchemokine.

(1) Preparation of Streptavidin-Conjugated AlkalinePhosphatase-Conjugated Biotinylated Oligosaccharide Fragment Derivedfrom any of Chondroitin Sulfate A, Chondroitin Sulfate C and ChondroitinSulfate E

Squid cartilage-derived chondroitin sulfate E (1 mg) was suspended in asolution (composition: 50 mM sodium acetate, 133 mM NaCl and 0.04%gelatin, pH 5.0) containing 0.6 mg of ovine testis hyaluronidase [1,800units; manufactured by Sigma]. The resulting reaction solution wasincubated at 37° C. for a total of 68.5 hours to obtain the digestionproduct containing a chondroitin sulfate E-derived oligosaccharidefragment. After 24 hours and 45.5 hours from initiation of incubation,additional 2 mg (6,000 units) of the enzyme was added to the reactionsolution.

In addition, each of whale cartilage-derived chondroitin sulfate A andshark cartilage-derived chondroitin sulfate C was suspended in asolution (composition: 50 mM sodium acetate, pH 5.0) containing 0.6 mgof ovine testis hyaluronidase [1,800 units; manufactured by Sigma]. Eachof the resulting reaction solutions was incubated at 37° C. for 24 hoursto obtain each of the digestion products containing a chondroitinsulfate A-derived oligosaccharide fragment and the digestion productcontaining a chondroitin sulfate C-derived oligosaccharide fragment.

Each of the resulting digest products was fractionated by HPLC with anamine-coupled silica PA-03 column using a linear gradient of 16 mM to 1MNaH₂PO₄. The resulting fractions were respectively subjected to SephadexG-25 column [1×30 cm; manufactured by Amersham-Biosciences] equilibratedwith distilled water. Elution was monitored by absorbance at 210 nm. Asa result, the fraction a, the fraction c, the fraction e-1 and thefraction e-2 shown in panel A of FIG. 7 were obtained.

(2) Analysis of Hydrocarbon Structure of Oligosaccharide Fragment

Each of the fragment a, the fragment c, the fragment e-1 and thefragment e-2 was digested with chondroitinase ACII (0.3 unit/ml) at 37°C. for 1 hour, and thereafter, the resulting digestion product wasderivatized with 2-AB. Then, each of the resulting products was analyzedby HPLC with amine-coupled silica PA-03 column with a linear gradientelution of 16 to 606 mM NaH₂PO₄ for 45 minutes.

As shown in panel B of FIG. 7, regarding a chodroitinase ACII-treatedfraction e-1, Di-di(4,6)S and ΔDi-4S were detected at a molar ratio ofabout 1:1. These peaks were shifted to Di-4S and ΔDi-4S positions,respectively, after treatment with a mixture of chondroitinase ACII andchondro-6-sulfatase, and further shifted to Di-0S and ΔDi-0S positions,respectively, after treatment with a mixture of chondroitinase ACII,chondro-6-sulfatase and chondro-4-sulfatase. Therefore, these resultssuggested that the fraction e-1 corresponds to the structure ofGlcAβ1-3GalNAc(4,6-O-disulfate)β1-4GlcAβ1-3GalNAc(4-O-sulfate). Suchstructure was also supported by mass spectrum.

Similarly, it was determined that the structure of the fraction acorresponds toGlcAβ1-3GalNAc(4-O-sulfate)β1-4GlcAβ1-3GalNAc(4-O-sulfate), that thestructure of the fraction c corresponds toGlcAβ1-3GalNAc(6-O-sulfate)β1-4GlcAβ1-3GalNAc(6-O-sulfate), and that thestructure of the fraction e-2 corresponds toGlcAβ1-3GalNAc(4,6-O-disulfate)β1-4GlcAβ1-3GalNAc(4,6-O-disulfate).

The above results are shown as a schematic view of an oligosaccharidestructure in panel C of FIG. 7. In panel C of FIG. 7, a solid triangledenotes GlcA, a hatched circle being GalNAc, 4S being 4-O-sulfation, 6Sbeing 6-O-sulfation, β3 being β1-3 linkage, and β4 being β1-4 linkage.

(3) Analysis of the Interaction Between Oligosaccharide Fragment and anyof L-Selectin, P-Selectin, CD44 and Chemokine

The interaction of an oligosaccharide contained in any of the fractiona, the fraction c, the fraction e-1 and the fraction e-2, with any ofL-selectin, P-selectin, CD44 and chemokine was examined.

First, each of oligosaccharides was biotinylated withbiotin-LC-hydrazide at a reducing end. To each of the fraction a, thefraction c, the fraction e-1 and the fraction e-2 obtained in the aboveitem (2), 125 mM EZ-link™ biotin-LC-hydrazide and 1M NaCNBH₃ in dimethylsulfoxide/acetic acid (7:3) were added. The resulting reaction mixturewas incubated at 65° C. for 3 hours, and then incubated at 37° C. for12.5 to 18.5 hours to biotinylate an oligosaccharide. Each of theresulting reaction products was subjected to Sephadex G-25 column asdescribed above, to remove unreacted biotin-LC-hydrazide, therebycollecting the fraction containing a biotinylated oligosaccharide. Then,the resulting fraction was evaporated to dryness.

A part of the resulting biotinylated oligosaccharide fraction wasdigested with chondrotinase ACII. Then, the resulting digestion productwas derivatized with 2-AB. As a result of HPLC analysis of the resultingproduct, a non-reducing end-derived disaccharide was detected ratherthan a reducing end-derived unsaturated disaccharide. Therefore, it wasmade clear that about 100% oligosaccharide was biotinylated.

Then, a part of a fraction containing 16 pmol of each biotinylatedoligosaccharide was dissolved in 0.5 ml of the buffer A, and thereafter,each of the resulting solutions and 1 μl (2 pmol) ofstreptavidin-conjugated alkaline phosphatase [manufactured by Promega]were incubated at 4° C. overnight.

Each of the resulting products was 3-fold diluted with the buffer Arespectively. Thereafter, the resulting solution was subjected to a wellof a 96 well flat bottom microtiter plate (Coster EIA/RIA plate number3690) coated with BSA (10 μg/ml), L-selectin-Ig (5 μg/ml), E-selectin-Ig(5 μg/ml), P-selectin-Ig (5 μg/ml), CD44-Ig (5 μg/ml), secondarylymphoid tissue chemokine (5 μg/ml), C-terminal truncated secondarylymphoid tissue chemokine (5 μg/ml), γ-interferon inducible protein-10(10 μg/ml), platelet factor 4 (2.5 μg/ml), stromal cell-derivedfactor/1β (5 μg/ml) or stromal cell-derived factor-1α (5 μg/ml). Bindingwas determined by measuring absorbance at 620 nm using Blue Phos™substrate. Results are shown in panel D of FIG. 7. In panel D of FIG. 7,expression of an abscissa axis is the same as that of FIG. 4. Inaddition, in each of lane, bar 1 denotes the results of the case wherestreptavidin-conjugated alkaline phosphatase is used, bar 2 being theresults of the case where biotinylated fraction a-conjugatedstreptavidin-conjugated alkaline phosphatase is used, bar 3 being theresults of the case where biotinylated fraction c-conjugatedstreptavidin-conjugated alkaline phosphatase is used, bar 4 being theresults of the case where biotinylated fraction e-1-conjugatedstreptavidin-conjugated alkaline phophatase is used, and bar 5 being theresults of the case where biotinylated fraction e-2-conjugatedstreptavidin-conjugated alkaline phosphatase is used.

As shown in panel D of FIG. 7, interestingly, only the e-2 fraction wasbound to L-selectin-Ig, P-selectin-Ig, secondary lymphoid tissuechemokine, γ-interferon inducible protein-10 and stromal cell-derivedfactor-1β, but the others did not bind thereto. The e-1 fractionmoderately bound to platelet factor 4, but did not bind to the otherchemokines, L-selectin or P-selectin. These results showed that arepeating GlcAβ1-3GalNAc(4-6-O-disulfate) unit is specificallyrecognized by L-selectin, P-selectin and many chemokines examined, andthat GlcAβ1-3GalNAc(4,6-O-disulfate) alone is probably sufficient forthe interaction with platelet factor 4.

Since each of L-selectin, P-selectin and chemokine is preferentiallybound to a tetrasaccharide composed of a repeatingGlcAβ1-3GalNAc(4,6-O-disulfate) unit as shown in FIG. 7, and since CD44interacts preferentially with an unsulfated chondroitin sulfate chain ora low-sulfated chondroitin sulfate chain as shown in Table 3, it isconsidered that, when a GlcAβ1-3GalNAc(4,6-O-disulfate) unit is presentas a cluster in glycosamanoglycan, probably, these units interact witheach of L-selectin, P-selectin and chemokine. In addition, it isconsidered that a different structure containingGlcAβ1-3GalNAc(4-O-sulfate) or GlcAβ1-3GalNAc(6-O-sulfate) may interactwith CD44.

EXAMPLE 8

Whether a persulfated CS/DS chain also inhibits chemokine activity ornot was examined. L1.2/CCR7 cells (1×10⁶ cells/ml) were loaded withFura-2, and then stimulated with secondary lymphoid tissue chemokine(“SLC” in FIG. 8) or C-terminal truncated secondary lymphoid tissuechemokine (“SLC-T” in FIG. 8), in the presence or absence ofglycosaminoglycan (100 μg/ml). The intracellular calcium concentrationwas monitored by measuring a fluorescent rate in the same manner asdescribed in the previous report [Hirose, J. et al., J. Biol. Chem.,276, 5228-5234 (2001)]. Chondroitin sulfate E (“CS E” in FIG. 8) andchondoroitin sulfate A (“CS A” in FIG. 8) were used as theglycosaminoglycan. The results are shown in FIG. 8. In FIG. 8, anarrowhead indicates a time point at which stimulation was given.

As shown in FIG. 8, secondary lymphoid tissue chemokine alone, orsecondary lymphoid tissue chemokine which had been pre-incubated withchondroitin sulfate A remarkably induced Ca²⁺ mobilization in L1.2 cellsin which a receptor of the secondary lymphoid tissue chemokine, CCR7 hadbeen incorporated by transfection, but secondary lymphoid tissuechmokine which had been pre-incubated with chondoroitin sulfate E didnot induce Ca²⁺ mobilization. Similarly, secondary lymphoid tissuechemokine which had been pre-incubated with chondoroitin polysulfate ordermatan polysulfate did not induce Ca²⁺ mobilization. On the otherhand, Ca²⁺ mobilization induced by C-terminal truncated secondarylymphoid tissue chemokine was not affetced by any of these persulfatedCS/DS chains. These results suggested that a persulfated CS/DS chaininhibits physiological activity of secondary lymphoid tissue chemokineby the interaction with a C-terminal region of secondary lymphoid tissuechemokine.

In addition, as shown in FIG. 8, since these glycosaminoglycans inhibitchemokine activity, it is thought that persulfated CS/DS-conjugatedchemokine may not function as an agonist for a chemokine receptor, butrather a persulfated CS/DS-chemokine complex may function as a reserverof chemokine in vivo. A low dissociation rate (2.78×10⁻⁴ to 5.30×10⁻³s⁻¹) observed in the interaction between chemokine and a persulfatedCS/DS chain supports this idea.

Sequence Listing Free Text

SEQ ID No.: 1 shows a sequence of a primer for amplifying CD44 gene.

SEQ ID No.: 2 shows a sequence of a primer for amplifying CD44 gene.

INDUSTRIAL APPLICABILITY

The saccharide compound of the present invention can be easily preparedupon preparation thereof. In addition, according to the saccharidecompound of the present invention, there can be achieved the regulationof the binding of any of L-selectin, P-selectin and chemokine to theligand thereof, the regulation of biological events mediated by any ofL-selectin, P-selectin and chemokine, improvement in symptom of adisease of which sideration is associated with the biological events,and provision of a lead compound for a therapeutic or prophylactic agentfor the disease. Further, the therapeutic or prophylactic agent of thepresent invention is useful for treating or preventing a disease ofwhich sideration is associated with biological events mediated by any ofL-selectin, P-selectin and chemokine, such as inflammatory disease,allergic disease, cancer metastasis, myocardial dysfunction, andmultiple organ failure.

1. A saccharide compound represented by the general formula (I):

wherein R¹, R², R³ and R⁴ each independently represent a hydrogen atomor a sulfonic group, or the general formula (II):

wherein R⁵, R⁶, R⁷ and R⁸ each independently represent a hydrogen atomor a sulfonic group.
 2. A saccharide compound represented by the generalformula (III):

wherein R⁹ and R¹⁰ each independently represent a hydrogen atom or asulfonic group, and m is 3 or 4, or the general formula (IV):

wherein R¹¹ and R¹² each independently represent a hydrogen atom or asulfonic group, and n is 3 or
 4. 3. A pharmaceutical compositioncomprising the saccharide compound of claim 1 or 2 as an activeingredient.
 4. An agent for treating or preventing a disease of whichsideration is associated with biological events mediated by any ofL-selectin, P-selectin and chemokine, wherein said agent comprises thesaccharide compound of claim 1 or 2 as an active ingredient.